The invention relates generally to regulators of G-protein signaling and more specifically to regulator of G-protein signaling 9 (RGS9) gene knockout animals and methods of using such animals to identify agents that regulate motor or other neurobehaviors. More specifically, the invention relates to methods of modulating dopamine D2 receptor signaling by an RGS protein, such as RGS9.
More than 20 mammalian members of the RGS family have been identified many of which are expressed in the brain (Siderovski and Strockbine et al. 1999; Zhang et al. 2000; Simonds and Zhang, 2000). The biological purpose of such multiplicity has remained unclear. These proteins may be involved in functions other than activating GTPase.
Many of the RGS proteins show a region specific expression in rat brain (Gold and Ni et al. 1997). A retina and a brain specific splice variant of RGS9 have been identified (Zhang and Howes et al. 1999). The long form of RGS9 (RGS9-2) is specifically expressed in the striatum (Thomas and Danielson et al. 1998; Rahman and Gold et al. 1999). RGS9 belongs to those members of the RGS family that have a Gxcex3-like domain and can form a complex with Gxcex2-proteins, especially Gxcex25 (Kovoor, Chen et al. 2000; Simonds and Zhang 2000). This complex seems to be essential for accelerating hydrolysis of Gxcex1t in rod outer segments since mice that do not express both alleles of RGS9 also lack Gxcex25 in retina (Chen, Burns et al. 2000).
RGS9 mRNA in striatum was detected in GABAergic medium spiny projection neurons (Thomas, Danielson et al. 1998). Medium spiny neurons in the striatum are believed to give rise to two distinct signaling pathways within the basal ganglia: the direct and the indirect pathway. The latter would originate in neurons that bear dopamine D2 receptors through which dopamine exerts inhibitory mechanisms. The direct pathway would originate in dopamine D1 receptor bearing medium spiny neurons where dopamine acts excitatory. Although this segregation of basal ganglia circuits remains disputed, it very well explains many of the anatomical and pharmacological findings related to dopamine signaling. The inhibitory action of dopamine D2 receptors is without doubt related to coupling to Gxcex1i (Picetti, Saiardi et al. 1997) whereas the excitation mediated via dopamine D1 receptors is coupled through Gxcex1s, Gxcex1o or Gxcex1z (Sidhu 1998)). Therefore it was very unlikely, that RGS9 was linked to dopamine D1 receptor, which interacts with Gxcex1s signaling.
Thus, there is a need for transgenic animals in which RGS9 is not expressed and a need to understand the role of RGS9 in dopamine D2-mediated signaling for modulating complex motor behavior.
A seminal discovery leading to the present invention is that an RGS protein, more specifically RGS9, modulates dopamine D2-mediated behavior. The present invention provides a transgenic non-human animal having a transgene disrupting or interfering with expression of RGS9, chromosomally integrated into germ cells of the animal and methods of using such transgenic animals to screen agents for modulation of dopamine D2-mediated behavior. The present invention also provides methods of modulating or treating dopamine D2-mediated behavior.
In a particular illustrative example, the invention provides a transgenic mouse including a disruption in the RGS9 gene. The disruption of the RGS9 gene results in an inability of the mouse to produce detectable levels of RGS9.
The invention also provides a method for producing a transgenic mouse exhibiting an inability to produce detectable levels of RGS9. The method includes introducing a transgene into a mouse embryonic stem cell, introducing the stem cell into a mouse embryo, transplanting the embryo into a pseudopregnant mouse and allowing the embryo to develop to term.
A method is also provided for screening a candidate agent for the ability to modulate dopamine D2-mediated behavior in a transgenic animal. The method includes administering to a first transgenic animal a candidate agent and comparing the dopamine D2-mediated behavior of the animal to the dopamine D2-mediated behavior of a second transgenic animal not administered the candidate agent.
A method is also provided for screening a candidate agent for the ability to modulate dopamine D2-mediated behavior. The method includes comparing the interaction between a RGS protein and a dopamine D2 receptor in the presence of a candidate agent and in the absence of a candidate agent, wherein a difference in the interaction between the RGS protein and the dopamine D2 receptor in the presence of the candidate agent compared to the interaction in the absence of the candidate is indicative of a candidate agent that modifies dopamine D2-mediated behavior.
A method is also provided for modulating a level of dopamine D2-mediated behavior. The method includes administering to a subject in need of such treatment an agent, wherein the agent modulates the activity of a RGS protein.
A method is also provided for modulating a locomoter response to a drug. The method includes administering to a subject in need of such treatment an agent, wherein the agent modulates the activity of a RGS protein.
A method is also provided for treating an abnormal motor behavior following a sensitization of dopamine D2 receptors. The method includes administering to a subject in need of such treatment an agent, wherein the agent increases the activity of a RGS protein.
A method is also provided for treating weight gain. The method includes administering to a subject in need of such treatment an agent, wherein the agent increases the activity of a RGS protein.